Intrinsic potential of phenotypically defined human hemopoietic stem cellsto self-renew in short-term in vitro cultures

In search for culture conditions that will facilitate hemopoietic stem cell (HSC) replication while preserving their primitive properties, we have mad e use of a multi-parameter FACS assay to define HSCs on basis of their phen otypic characteristics, i.e., CD34(++)CD33,38,71(-). Bone marrow and umbili cal cord blood samples of CD34(+) cells from 31 donors were loaded with the membrane dye PKH26 and each exposed to various culture conditions for 6 da ys. The cells that retained the primitive CD34(++)CD33,38,71(-) phenotype w ere analysed for the number of cell replications they underwent, by measuri ng loss of PKH26 fluorescence after 6 days. A most striking observation was the large inter-sample variation in the proliferative response of cells th at retained the CD34(++)CD33,38,71(-) phenotype. In general, samples could be characterised as either good- or poorly-replicating, according to the pr oliferation property of their CD34(++)CD33,38,71(-) subset. In comparison t o this 'intrinsic' potential, the effects of the applied growth stimuli on CD34(++)CD33,38,71(-) cell replication were negligible. In contrast, the ov erall recovery of the CD34(++)CD33,38,71(-) cells was clearly dependent on the culture stimuli. Of the various conditions tested, serum-free cultures with pre-established stroma maintained the cells with this primitive phenot ype most effectively. In cultures supplemented with various combinations of recombinant HGFs, HSC differentiation prevailed. These findings with pheno typically defined HSCs should assist in the design of systems for expansion and ex vivo gene therapy of early hemopoietic cells. (C) 1999 Internationa l Society for Experimental Hematology. Published by Elsevier Science Inc.